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Abstract

Introduction

Cell therapy is a potential therapeutic approach for neurodegenerative disorders,
such as Alzheimer disease (AD). Neuronal differentiation of stem cells before transplantation
is a promising procedure for cell therapy. However, the therapeutic impact and mechanisms
of action of neuron-like cells differentiated from human umbilical cord mesenchymal
stem cells in AD have not been determined.

Methods

In this study, we used tricyclodecan-9-yl-xanthogenate (D609) to induce human mesenchymal
stem cells isolated from Wharton jelly of the umbilical cord (HUMSCs) to differentiate
into neuron-like cells (HUMSC-NCs), and transplanted the HUMSC-NCs into an AβPP/PS1
transgenic AD mouse model. The effects of HUMSC-NC transplantation on the cognitive
function, synapsin I level, amyloid β-peptides (Aβ) deposition, and microglial function
of the mice were investigated.

Results

We found that transplantation of HUMSC-NCs into AβPP/PS1 mice improved the cognitive
function, increased synapsin I level, and significantly reduced Aβ deposition in the
mice. The beneficial effects were associated with “alternatively activated” microglia
(M2-like microglia). In the mice transplanted with HUMSC-NCs, M2-like microglial activation
was significantly increased, and the expression of antiinflammatory cytokine associated
with M2-like microglia, interleukin-4 (IL-4), was also increased, whereas the expression
of proinflammatory cytokines associated with classic microglia (M1-like microglia),
including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), was significantly
reduced. Moreover, the expression of Aβ-degrading factors, insulin-degrading enzyme
(IDE) and neprilysin (NEP), was increased substantially in the mice treated with HUMSC-NCs.

Keywords:

Introduction

Alzheimer disease (AD) is an age-related progressive neurodegenerative disorder. The
major symptoms of AD include memory loss and severe cognitive decline. Pathology of
the AD brain is characterized by amyloid plaques, neurofibrillary tangles, and neuronal
loss. Elevated amyloid β-peptide (Aβ) deposition is the key pathogenic factor for
AD and the main cause for neuronal loss in AD [1]. Thus, promising therapeutic strategies for AD aim to prevent, reverse, and reduce
Aβ deposition [2].

Cell therapy is a potential therapeutic approach for neurodegenerative disorders,
such as AD [3,4]. It has been found that transplantation of cells isolated from human umbilical cord,
mesenchymal stem cells (MSCs), or neural progenitor cells improves neuropathology
in animal models of AD through modulation of neuroinflammation [5-8]. Recently, a number of studies demonstrated that transplantation of neuronal cells
induced from MSCs produces beneficial effects in neurodegenerative diseases and spinal
cord injury [9-11]. It has been shown that neuronal cells differentiated from human MSCs are more resistant
to Aβ42 oligomer-induced cytotoxicity than are undifferentiated cells [12]. Thus, neuronal differentiation before transplantation could yield better efficacy
than transplantation of undifferentiated MSCs in clinical application.

The mechanism underlying the beneficial effects of stem cell transplantation on neurodegenerative
diseases has been found to be associated with microglial function in brain. Microglia,
the mononuclear phagocytes of brain, accumulate in senile plaques in AD patients and
in animal models of AD. It has been shown that microglia release proinflammatory cytokines,
such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β),
and NO, causing neurodegeneration [13]. In contrast, growing evidence has also supported that “alternatively activated”
microglia (M2-like microglia) play protective roles in AD by phagocytizing Aβ and
secreting neurotrophic cytokines [14]. Multiple studies demonstrated that intracerebral transplantation of MSCs increases
M2-like microglial activation, regulates neuroinflammation, and reduces Aβ deposits
in AD mouse models [8,9,15].

In this study, we used tricyclodecan-9-yl-xanthogenate (D609) to induce HUMSCs to
differentiate into neuron-like cells (HUMSC-NCs) and transplanted the HUMSC-NCs into
an AβPPswe/PS1dE9 mouse model of AD. We found that transplantation of HUMSC-NCs reduced
Aβ deposition in the mouse and improved the mouse’s cognitive function. The beneficial
effects might be associated with M2-like microglial activation and modulation of neuroinflammation
by HUMSC-NC transplantation.

Materials and methods

Animals

The heterozygous AβPPswe/PS1dE9 double-transgenic mouse with C57BL/6 background was
used in this study. The mouse harbors the mutant human genes APPswe (Swedish mutations
K594N/M595L) and presenilin-1 with the exon-9 deletion (PS1-dE9) under the control of mouse prion protein promoter. This type of transgenic mouse
has been used widely in the study of AD [16,17]. The mouse shows typical characteristics of AD and exhibits an early appearance of
amyloid plaques deposition and increased proinflammatory microglial activation [17,18]. Because of the gender-specific differences in the progression of amyloid plaque
deposition, we used only male mice in this study. The mice were obtained from Beijing
HFK Bio-Technology Co., Ltd., Institute of Laboratory Animal Science, Chinese Academy
of Medical Science (Beijing, China), and housed in temperature- and humidity-controlled
rooms on a 12 hour/12 hour light/dark cycle. Breeder mice carrying the transgenes
are backcrossed to the C57BL/6 strain for six to seven generations to obtain mice
used in research. The mice are genotyped to confirm the presence of the mutant genes
by polymerase chain reaction (PCR) amplification of genomic DNA extracted from tail
clippings before the mice are sold to research laboratories. All the procedures described
in this study were in accordance with the Ethical Committee for Animal Experiments
of Shandong University.

HUMSC isolation and in vitro culture

Human umbilical cords were obtained from full-term deliveries with the informed consent
from parents after caesarian section. The procedure for collecting tissues was approved
by the ethical committee of the Second Hospital of Shandong University. The procedure
was based on the previous description by Huang et al.[19]. After arteries and veins were removed, the remaining tissue, Wharton jelly, was
cut into 0.5- to 1-cm3 pieces and suspended in Dulbecco modified Eagle low-glucose media (DMEM-LG; Gibco,
Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco), 100 mg/ml penicillin,
and 100 mg/ml streptomycin. The tissue was left undisturbed for 7 days in a 37°C humidified
incubator with 5% CO2 to allow cells to migrate from the explants. Culture media was replaced every 3 days.
The cells were passaged by using 0.25% trypsin-EDTA solution when they reached 80%
to 90% confluence. The cells were analyzed with flow cytometry to confirm the immune-phenotype
of HUMSCs according to the previous report [20]. The cells used in this study were positive for CD73, CD90, and CD105, but negative
for CD34, CD45, and HLA-DR, consistent with HUMSC characteristics.

Induction of neuron-like cells from HUMSCs

After HUMSCs were passaged 2 to 6 times and reached subconfluence, the cells were
washed twice with basal DMEM media (without FBS) and divided into two groups. In the
control group, the cells were cultured in basal DMEM medium (without FBS); in the
D609 treatment group, the cells were cultured in basal media containing 60 μg/ml D609
(J&K, Beijing, China) to induce neuronal differentiation. D609 was prepared freshly
in deionized water. We determined the optimal working concentration of D609 by performing
a dose response of HUMSCs to 2 to 100 μg/ml of D609, and we found that 60 μg/ml D609
was the optimal concentration for neuronal differentiation (data not shown). The morphology
of cells was observed under a phase-contrast microscope.

Immunocytochemistry

To determine the expression of neuronal cell markers, HUMSCs grown on glass coverslips
were treated with D609 or basal medium for 6 hours. The cells were fixed in 4% paraformaldehyde
in phosphate-buffered saline (PBS) for 20 minutes and then washed 3 times with PBS.
The cells were blocked with normal goat serum for 20 minutes at room temperature and
then incubated in the following primary antibodies: neuron-specific enolase (NSE,
rabbit IgG, 1:200, Abcam, Cambridge, MA, USA), microtubule-associated protein 2 (MAP2,
rabbit IgG, 1:200, Abcam), and glial fibrillary acidic protein (GFAP; rabbit IgG,
1:200, Sigma, St Louis, MO, USA). The cells were then washed in PBS and incubated
with the secondary antibodies (goat anti-rabbit IgG-TRITC) for 1 hour at room temperature.
After washing with PBS, the cells were observed under a fluorescence microscope (Olympus
1 × 71S1F-3, Tokyo, Japan). The differentiation rate of HUMSCs was calculated according
to the following formula: The differentiation rate (percentage) = (the number of NSE
or MAP2 positive cells/the total number of cells) ×100. For each sample, 10 randomly
selected visual fields were used for the calculation. The results presented are the
mean ± SEM from three independent experiments.

Transplantation of HUMSC-NCs in AβPP/PS1 double-transgenic mice

HUMSC-NC suspension, HUMSC suspension, or PBS was injected into 6-month-old male AβPP/PS1
transgenic mice for only one time. Before the injection, the cell suspension was washed
with PBS for 3 times to remove drug and serum. Animals were anesthetized with 10%
chloral hydrate and placed in a small animal stereotaxic apparatus with a microinjector
unit. Either cell suspension or PBS was injected into mouse bilateral hippocampus
at the following coordinates: 1.6 mm posterior to the bregma, 1.7 mm bilateral to
the midline, and 2.5 mm ventral to the skull surface. Cell suspension was injected
with a 25-μl syringe. Then 2 μl of cell suspension (approximately 5 × 104 cells) was injected into the hippocampus bilaterally. Cell suspension or PBS was
delivered at a rate of 0.3 μl/min. After injection, the needle was left in place for
5 minutes before being retracted.

Behavior test

Three weeks after transplantation, we used the modified Morris water-maze test to
assess spatial memory performance [21]. The procedure consisted of 1 day of adapting tests without platform and 5 days of
hidden-platform tests, plus a spatial probe test 24 hours after the last hidden-platform
test; 15 mice were used in each group. The detailed method was described in our previous
report [22]. In brief, in the spatial-acquisition tests, mice were released into the pool and
given 60 seconds to find the hidden platform. If a mouse did not find the platform
within 60 seconds, it was guided to the platform. Animals were given four trials per
day. The distal starting positions were semirandomly selected. For basic acquisition
training, the platform was located in the southwest quadrant. The starting positions
were north, east, southeast, and northwest. The time to find the platform was recorded
as the latency for each trial. A single probe trial, in which the platform was removed,
was performed after the hidden-platform task had been completed. Mice were placed
in a novel start position (northeast) in the maze, and each mouse was allowed to swim
for 60 seconds. The data were analyzed with multivariate analysis of variance (ANOVA).
Mouse behavior was observed blindly. Observers were kept ignorant of the treatment
given to each animal group.

Thioflavin S staining and immunohistochemical staining

AβPP/PS1 mice were killed after the behavior test. The mice were anesthetized with
chloral hydrate, and then were immediately cardiac perfused with 0.9% saline solution
followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). After the perfusion, the brains of the mice were excised and postfixed
overnight at 4°C. The brain tissue was then incubated in 30% sucrose at 4°C until
equilibration (six mice per group). Then 30- or 10-μm coronal sections were cut with
a freezing microtome (Leica CM1850, Leica Microsystems, Heidelberg Germany) and stored
at −20°C.

Thioflavin S staining was performed on floating sections (30-μm thickness). Brain
sections were incubated in 0.5% thioflavin S (Sigma-Aldrich, USA) dissolved in 50%
ethanol for 5 minutes, and then washed twice with 50% ethanol for 5 minutes each time.
The brain sections were washed once with tap water for 5 minutes, and then mounted
with mounting medium [8]. The green fluorescence-stained plaques were observed under a fluorescence microscope.
Frontal cortex, cingulate, and hippocampus were examined for amyloid load. According
to the previous report [23], these regions have plaque prevalence in AD patients and are involved in memory functions.

Quantitative real-time PCR

Total RNA from cortex and hippocampus of one hemisphere was extracted by using Trizol
(Invitrogen) according to the manufacturer’s protocol (six mice per group); 1 μg total
RNA was used for reverse transcription in a final volume of 20 μl. Reverse transcription
was performed according to the manufacturer’s protocol (DRR047A, Takara, Otsu, Shiga,
Japan). Then 2 μl cDNA was used for real-time PCR with the SYBR Premix Ex Taq (DRR041A,
Takara, Japan). Quantitative real-time PCR was performed under the following conditions:
95°C for 30 seconds, 95°C for 5 seconds, 60°C for 34 seconds, and 40 cycles. All PCR
reactions were performed in triplicate. The fold changes of target genes were calculated
by using the delta delta cycle threshold (two delta delta CT) method [24]. GAPDH was used as the reference gene. The PCR primers used in the study were reported
previously [8].

Statistical analysis

All data were presented as mean ± standard error of the mean (SEM). Differences were
analyzed with ANOVA. A value of P < 0.05 was considered statistically significant. In two-variable experiments, two-way
ANOVA followed by Bonferroni post hoc tests were used to analyze the significance of differences between groups. One-way
ANOVA was used to analyze one-variable experiments with three groups. The Student
t test was used to compare two groups. Data were analyzed with the software SPSS 16.0
(SPSS Inc., Chicago, IL, USA).

Results

D609 induces neuron-like differentiation of HUMSCs

Wang and colleagues found that D609 can induce rat MSCs and human bone marrow stem
cells to differentiate into neuron-like cells within 6 hours. Thus, we examined the
morphologic change and the expression of neuronal markers of HUMSCs after 6-hour treatment
with D609. HUMSCs appeared as large and flat cells in the absence of D609 treatment.
After 6 hours of D609 treatment, most of the cells exhibited a typical neuron-like
morphology characterized by a multipolar cell body with peripheral extended processes,
and the processes formed a connected network; no obvious morphologic change was found
in the control group (HUMSCs, Figure 1A). Immunocytochemistry analysis showed that HUMSC-NCs expressed high levels of the
neuronal cell markers, neuron-specific enolase (NSE) and microtubule-associated protein
2 (MAP2) (Figure 1B). The percentage of cells expressing NSE or MAP2 was 86.42% ± 6.787% or 82.39% ±
5.644% in the D609-treated group. However, in the control groups, HUMSCs expressed
NSE or MAP2 weakly (Figure 1B). To examine further the quality and quantity of the neuronal differentiation, we
examined the expressions of glial fibrillary acidic protein (GFAP), a marker for glial
cells, in the D609-induced cells. We did not find GFAP expression in the differentiated
cells, indicating that D609 induces HUMSCs to differentiate into neuron-like cells
but not glial cells (see Additional file 1: Figure S1).

To determine the effect of HUMSC-NC transplantation on the behavior of AβPP/PS1 mice,
we used a Morris water maze to examine the spatial learning and memory of the mice
3 weeks after they received HUMSC-NC, HUMSC, or PBS injection. No difference in behavioral
performance was detected in the HUMSC-treated mice compared with the PBS-treated mice.
However, the mice treated with HUMSC-NCs performed significantly better in the water-maze
test than did the mice treated with PBS. The mean escape latency of the mice treated
with HUMSC-NCs was significantly shorter than that of the mice treated with PBS (2A).
We evaluated mouse spatial memory by performing probe trials 24 hours after the last
training session. The number of platform location crosses and the time spent in the
target quadrant of the mice treated with HUMSC-NCs was significantly higher and longer
than were those of the mice treated with PBS (Figure 2B, C). The swimming speed was not significantly different among the three groups (Figure 2D), suggesting that the improved behavioral performance of the mice treated with HUMSC-NCs
was caused by cognitive processes but not noncognitive components of behavior. Thus,
our data suggest that single intracerebral injection of HUMSC-NCs alleviates learning
deficits and memory impairments in AD mice, whereas single injection of HUMSCs does
not.

HUMSC-NC transplantation increases synapsin I level

The cognitive impairment in AD is associated mostly with synaptic loss and dysfunction.
Synapsin I has been shown to play a key role in functional maturation of synapse and
neurotransmitter release [25]. We then compared the expression of synapsin 1 protein in the hippocampus between
HUMSC-NC-transplanted and PBS-treated mice. We found that synapsin I protein expression
in hippocampus was increased significantly in HUMSC-NC-transplanted mice compared
with that in PBS-treated mice (Figure 3), suggesting that the cognitive improvement in HUMSC-NC-treated mice might be associated
with the upregulation of synapsin 1.

Figure 3.HUMSC-NC transplantation increased synapsin I level. (A) Representative Western blots for synapsin I protein expression in hippocampus. (B) Quantification of the Western blot for synapsin I. The densitometry of synapsin I
bands was first normalized to the loading control β-actin. The percentage of the expressions
of synapsin I in the HUMSC- or HUMSC-NC-treated group relative to that in the PBS-treated
group was then calculated. The scanned image of Western blot was analyzed with the
software Image J. Data were presented as mean ± SEM. *P < 0.05, HUMSC-NC-treated group versus PBS-treated group.

Aβ deposition is the key pathogenic factor for AD and causes cognitive deficits. We
investigated the effect of HUMCS-NC transplantation on Aβ deposition in AD mice. We
used thioflavin S staining to determine Aβ deposition. We found that Aβ deposition
in both the cortex and hippocampus of the mice treated with HUMSC-NCs was dramatically
reduced compared with that of the mice treated with PBS (Figure 4A, B). Because Aβ can be released by cleavage of a99-residue C-terminal fragment (CTF),
we measured the CTF level in the cortex or hippocampus of the mice with Western blot.
No difference in the CTF level was found among the three groups (data not shown),
indicating that HUMSC-NC transplantation does not affect Aβ production. Further to
confirm the reduction of Aβ deposition by HUMSC-NC transplantation, we performed ELISA
to quantify soluble Aβ40 and Aβ42 levels in the mice. We found that both Aβ40 and Aβ42 levels in the mice treated with HUMSC-NCs were significantly decreased compared with
those in the mice treated with PBS (Figure 4C, D). Our results suggest that HUMSC-NC transplantation significantly reduces Aβ
deposition in the AD mice.

Figure 4.HUMSC-NC transplantation reduced Aβ deposition and soluble Aβ levels. (A) Thioflavin S staining for senile plaques of cerebral cortex and hippocampus in the
mice. The staining was performed according to the description in the Methods section.
Images were captured with a camera system connected to a fluorescence microscope (Olympus
1×71S1F-3, Japan). Scale bar, 200 μm. (B) Quantification of thioflavin S staining. The plaque burden was calculated as the
percentage of thioflavin S staining area over the total area (n = 6 in each group). Image Pro Plus 6 (Media Cybernetics) was used to analyze the
images. (C, D) ELISA for soluble Aβ40(C) and Aβ42(D) in the cortex and hippocampus. Mouse brain tissue was homogenized, and soluble protein
was extracted. Aβ ELISA kits (Invitrogen) was used to determine the levels of Aβ40 and Aβ42 (n = 6 in each group). Data are presented as mean ± SEM; *P < 0.05; **P < 0.01; HUMSC-NC-treated group versus PBS-treated group.

We then further investigated the mechanism by which HUMSC-NC transplantation reduces
Aβ deposition. In our study, we discovered that HUMSC-NC transplantation significantly
increased the number of activated microglia in the cortex and hippocampus of the mice
compared with PBS injection (Figure 5A, B). The Iba1 staining showed that the microglia in the mice treated with HUMSC-NCs
displayed an activated microglial morphology characterized by large cell body and
thick processes (Figure 5A). The density of the activated microglia reached the highest around the plaques,
corpus callosum, hippocampus, and cortex area near the injection site (data not show).
Microglial activation was not observed in the mice treated either with HUMSCs or with
PBS injection. These data indicate that HUMSC-NC transplantation stimulates microglial
activation in AβPP/PS1 mice.

Figure 5.HUMSC-NC transplantation increased microglial activation. (A) Immunofluorescence staining for microglia with anti-Iba1 in both the hippocampus
and cortex. The staining was performed as described in the Methods section. The processed
tissue section was stained with rabbit anti-mouse Iba-1 IgG (1:500, Wako), and then
visualized with FITC-conjugated secondary antibody. Scale bar represents 20 μm. (B) Quantification of the staining images. Iba-1 burden was calculated as the percentage
of Iba-1-positive area over the total area. The image quantification was performed
by using the software Image Pro Plus 6 (Media Cybernetics). Data are presented as
mean ± SEM. *P < 0.05; **P < 0.01; HUMSC-NC-treated group versus PBS-treated group.

Activated microglia can be either neuroprotective or neurodestructive, depending on
the phase of activation. Alternatively, activated microglia (M2-like microglia) have
been found to protect neurons by increasing Aβ clearance and reducing neuroinflammation.
Thus, we used the following markers for M2-like microglia: Chitinase 3-like 3 (YM-1),
arginase-1 (Arg-1), AMCase, mannose receptors C type 1 (MRC1), found in inflammatory
zone 1 (FIZZ1), and haptoglobin/hemoglobin scavenger receptor (CD163) to examine whether
the activated microglia induced by HUMSC-NC transplantation were M2-like microglia.
Our qRT-PCR assay showed that the gene-expression levels of YM-1, Arg-1, MRC1, FIZZ1,
and CD163 in both the cortex and hippocampus of the mice treated with HUMSC-NCs were
increased substantially, compared with those in the mice treated with PBS (Figure 6A). Our immunofluorescence staining also demonstrated that AMCase expression was colocalized
with Iba-1expression in microglia (Figure 6B), indicating that the activated microglia induced by HUMSC-NC transplantation have
M2-like microglial characteristics. Our results suggest that HUMSC-NC transplantation
induces M2-like microglial activation in AD mice.

Figure 6.HUMSC-NC transplantation induced M2-like microglial activation and modulated neuroinflammation.
(A) mRNA levels of Arg-1, YM-1, MRC1, FIZZ1, and CD163 in the cortex or hippocampus were
determined by quantitative RT-PCR. GAPDH was used as the reference gene. (n = 6 in each group). (B) Immunofluorescence staining for AMCase and Iba-1. Tissue sections were processed
and stained with rabbit anti mouse Iba-1 IgG (1:500; Wako) and goat anti-mouse AMCase
IgG (1:200, Santa Cruz). FITC- or TRITC-conjugated secondary antibodies (1:200) were
used to visualize the staining. Scale bar represents 50 μm. (C) mRNA levels of IL-4, TNF-α, IL-1β, and IL-6 in the cortex or hippocampus were measured
with quantitative RT-PCR (n = 6 in each group). (D) Double immunofluorescence staining for TNF-α and Iba-1 in the cortex and hippocampus
area. (E) Double immunofluorescence staining for IL-4 and Iba-1 in the cortex and hippocampus
area. The images were merged from individual images with different staining. Images
were captured by a camera system connected to the fluorescence microscope (Nikon Eclipse
90i LH-M100CB-1). Scale bar represents 20 μm. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; HUMSC-NC-treated group versus PBS-treated group.

AβPPswe/PS1dE mice have significantly higher levels of proinflammatory cytokines,
including IL-1β, IL-6, and TNF-α, than do wild-type mice [18]. Reducing these cytokines has been shown to prevent neuronal dysfunction in AD [26]. We examined whether the beneficial effects of HUMSC-NC transplantation were associated
with a reduction of the proinflammatory factors IL-1β, TNF-α, and IL-6. Our RT-PCR
result showed that the mRNA levels of IL-1β and TNF-α were significantly reduced in
the cortex and hippocampus of the mice treated with HUMSC-NCs compared with those
in the mice treated with PBS (Figure 6C), whereas the IL-6 level was not different between the two groups. M2-like microglial
activation has been shown to be associated with upregulation of the antiinflammatory
cytokine, IL-4 [27]. Our RT-PCR result showed that the mRNA level of IL-4 in the cortex and hippocampus
of the mice treated with HUMSC-NCs was significantly increased compared with that
of the control (Figure 6C).

To determine further whether the downregulation of the proinflammatory cytokine TNF-α
and the upregulation of the antiinflammatory cytokine IL-4 were related to the activated
microglia in the mice treated with HUMSC-NCs, we performed double immunofluorescence
staining for TNF-α and Iba-1, or IL-4 and Iba-1. Significant increase of IL-4 expression
and decrease of TNF-α expression were detected in the Iba-1-positive cells in the
cortex and hippocampus of the mice treated with HUMSC-NCs compared with those in the
control (Figure 6D, E). This result suggests that M2-like microglial activation is associated with
the modulation of neuroinflammation in the AD mice.

The expression of Aβ-degrading factors is stimulated by HUMSC-NC transplantation

Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the most important Aβ-degrading
enzymes. Our RT-PCR result demonstrated that the mRNA levels of NEP and IDE in the
mice treated with HUMSC-NCs were significantly higher than those in the mice treated
with PBS (Figure 7A, B). IDE protein level in both the cortex and hippocampus was also significantly
increased by HUMSC-NC transplantation (Figure 7C, E), whereas NEP protein level was significantly elevated in the hippocampus but
not in the cortex of the mice treated with HUMSC-NCs (Figure 7C, D) compared with that in the control. Our results indicate that HUMSC-NC transplantation
reduces Aβ deposition in AD mice by increasing Aβ removal.

Figure 7.HUMSC-NC transplantation increased the expression of Aβ-degrading enzymes NEP and
IDE. (A, B) The mRNA levels of NEP (A) and IDE (B) in the cortex or hippocampus were determined with RT-PCR. Both NEP and IDE mRNA levels
were increased significantly in the cortex and hippocampus of HUMSC-NC-treated mice.
(C) Representative Western blots for NEP and IDE protein expression in the cortex or
hippocampus: 100 μg of protein was load in each lane. Duplicate samples of each group
were loaded into the gel. (D, E) Quantification of the Western blot for NEP (D) and IDE (E). The densitometry of NEP and IDE bands were first normalized to the loading control
β-actin. The percentage of the expressions of NEP and IDE in the HUMSC- or HUMSC-NC-treated
group relative to that in the PBS-treated group was then calculated. The scanned image
of Western blot was analyzed with the software Image J. Data were presented as mean
± SEM. *P < 0.05, HUMSC-NC-treated group versus PBS-treated group.

Discussion

In this study, we used D609 to induce HUMSCs to differentiate into HUMSC-NCs and transplanted
the HUMSC-NCs into AβPP/PS1 mice. We found that HUMSC-NC transplantation reduced Aβ
deposition and alleviated cognitive decline through a mechanism associated with activating
M2-like microglia and upregulating neuroprotective cytokines.

D609 has been shown to increase markedly the expression level and activity of molecules
associated with the oxidative state of MSCs, including reactive oxygen species (ROS),
NADPH oxidase, and Rb protein [28]. The possible mechanism underlying D609-induced neuronal differentiation of HUMSCs
might be associated with the changes of these molecules.

The mice were not provided immunosuppression. HUMSCs have been found to be able to
escape immune surveillance, possibly because of the absence of the antigen major histocompatibility
complex II (MHC-II) and the co-stimulatory surface antigens CD40, CD80, and CD86 [29-31]. It has also been shown that neuron-like cells differentiated from human MSCs do
not express CD40 and CD86 [32]. These findings suggest that both HUMSCs and the neuron-like cells differentiated
from human MSCs might have low immunogenicity and might be well tolerated in xenotransplantation
without immunosuppression. Consistent with our findings, a number of studies have
demonstrated that transplantation of human MSCs or neuron-like cells differentiated
from MSCs without immunosuppression produces beneficial effects in animal models of
AD, Parkinson disease, traumatic brain injury, and stroke [10,33-36].

The beneficial effects produced by HUMSC-NC transplantation might not directly come
from the HUMSC-NCs, but instead could be indirect effects caused by the transplanted
cells. We found injected cells in the hippocampus and cortex of the mice 1 week after
the transplantation (data not shown); however, 1 month after the transplantation,
few transplanted cells were detected, both in the mice treated with HUMSCs and in
the mice treated with HUMSC-NCs. Similar observations have been reported by other
research groups. Hong et al.[33] and Parr et al.[37] showed that only small numbers of transplanted stem cells or induced neuron-like
cells engraft into injured tissues, and only a few grafted cells survive. It has been
proposed that the beneficial outcome of cell transplantation in neurodegenerative
diseases could result from the paracrine factors induced by cell transplantation and
be sustained by the paracrine factors even after transplanted cells die [7,38]. Our results supported such hypotheses. We found that the expression of antiinflammatory
cytokine IL-4 was significantly increased by HUMSC-NC transplantation, which could
lead to the induction of M2-like microglial activation and Aβ removal in the AD mice.

NEP and IDE are the most important Aβ-degrading enzymes. We found that HUMSC-NC transplantation
significantly increased IDE and NEP expression in the AD mice. Deletion of the NEP or IDE gene in mice increases cerebral Aβ accumulation, whereas overexpression of NEP and
IDE reduces Aβ levels [39-41]. Multiple studies showed that microglia secretes IDE and NEP [8,42,43]. Antiinflammatory cytokine IL-4 has also been found to increase IDE and NEP levels,
both in vitro and in vivo[44,45]. In our study, we observed both M2-like microglial activation and upregulation of
IL-4 expression in the mice treated with HUMSC-NCs, which then could lead to the increase
of NEP and IDE expression. We observed an elevation of both mRNA and protein levels
of NEP and IDE by HUMSC-NC transplantation. Our results suggest that HUMSC-NC transplantation
reduces Aβ deposit by elevating Aβ degradation.

M2-like microglial activation is stimulated in an AD mouse model when MSCs are transplanted
into the mice [8]. In our study, single intracerebral injection of neuron-like cells differentiated
from HUMSCs into a similar AD mouse model also significantly promoted M2-like microglial
activation. M2-like microglia have been shown to play protective roles in AD through
the following three mechanisms: promoting Aβ clearance by directly secreting enzymes,
phagocytizing Aβ, and secreting neuroprotective cytokines [8,46-48].

The mechanism underlying M2-like microglial activation by cell transplantation remains
unclear. It could be an indirect effect from transplanted cells. We proposed that
D609-induced neuron-like cells could stimulate the secretion of some bioactive paracrine
factors, which then promote M2-like microglial activation. It has also been shown
that M2-like microglia can be recruited from bone marrow in an AD mouse model [7,49,50]. Thus, in our study, the source of M2-like microglia activated by HUMSC-NC transplantation
could be either from bone marrow recruitment or from local resting microglia that
could be activated into M2-like microglia by paracrine factors stimulated by HUMSC-NC
transplantation, such as IL-4.

Antiinflammatory cytokine IL-4 has been found to promote M2-like microglial activation
[51] and induces Aβ removal both in vitro and in vivo[44,45,52]. We observed an upregulation of IL-4 expression by HUMSC-NC transplantation, and
our immunofluorescence staining showed that IL-4 expression was markedly increased
in Iba-1-positive cells in particular. Our results suggest that M2-like microglial
activation in the mice treated with HUMSC-NCs could be mediated by upregulation of
IL-4 expression.

Both our RT-PCR and immunocytochemistry staining results suggest that HUMSC-NC transplantation
downregulates the expression of proinflammatory cytokines, including TNF-α and IL-1β.
TNF-α, IL-6, and IL-1β are markers for classic microglia (M1-like microglia). These
proinflammatory cytokines have been shown to promote Aβ production and reduce Aβ removal
in an AD mouse model [53]. Aβ-induced phagocytosis of microglia can be inhibited by proinflammatory cytokines,
such as IL-1β, TNF-α, IFN-γ, MCP-1, and CD40L [54]. Thus, reducing these proinflammatory cytokines could promote Aβ clearance in AD.
Our results suggest that HUMSC-NC transplantation reduces the chronic inflammation
mediated by M1-like microglia and induces M2-like microglial activation to enhance
Aβ removal, which consequently results in cognitive improvement in the AD mice.

In our study, HUMSC transplantation did not produce beneficial effects in the AD mice;
although transplantation of MSCs has been shown to rescue memory deficits and reduce
Aβ deposition in a similar AD mouse model [8]. The inconsistent observation might be because different experimental procedures
for cell transplantation were used in the studies. In our study, we performed single
injections of HUMSCs into mice, whereas in the studies of others, mice received double
or triple injections of MSCs. In addition, it has been shown that a single injection
of bone-marrow MSCs does not promote M2-like microglial activation and fails to improve
the cognitive activity in an AD mouse model, whereas multiple transplantations produce
beneficial effects [8]. We found that single transplantation of HUMSC-NCs improved memory and reduced Aβ
deposition in the AD mice, which is consistent with others’ observation that single
transplantation of neuron-like cells differentiated from MSCs produces beneficial
effects in neurodegenerative diseases and spinal cord injury [9-11].

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HY designed and performed the experiments and wrote the manuscript. ZHX participated
in designing the experiments. LFW, HNY, and SNY provided assistance for data analysis,
mouse-injection experiments, and ELISA assay, respectively. ZYZ, PW, and CPZ were
responsible for mouse-behavior observation. JZB participated in designing the experiments
and drafting the manuscript. All authors read and approved the manuscript for publication.

Acknowledgements

This research was supported by National Basic Research Program of China (2009CB526507),
the National Natural Science Foundation of China (81171214), and Jinan Science and
Technology Development Foundation (200906182–2).

References

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